The following specification particularly describes the nature of the invention and the manner in which it is to be performed:-

t OBjlCIlIPTIQN

^ALCOIIOL.G.
Technical Field
This invention relates to a process for producing
optically active j3 -amino alcohols. More particularly, it relates to a process for producing optically active
j3- -amino alcohols which are of value as drugs or their
intermediates.
Background Art
Ephedrines have been used for purposes of perspiration, antipyresis and cough soothing from the olden times, and particularly, d-pseudoephedrine is known to possess anti-inflammatory action. Pharmacological action such as vasoconstriction, blood pressure elevation, or perspiration is known for 1-ephedrine and it is used in therapy as a sympathomimetic agent. 1-Ephedrine is also used in the treatment of bronchial asthma. Specifically, processes for the production of optically active j3 -amino alcohols, including optically active ephedrines, are useful in the manufacture of drugs and their intermediates; thus, there is a need for efficient production processes.
In the conventional process for, producing a j3 -amino alcohol with the desired optical'activity, there

was used a process by which a racemic j3 -amino alcohol is obtained and then a specific optically active form is produced by optical resolution or asymmetric synthesis among others.
However, since the racemic j3 -amino alcohol has two asymmetric carbons within its molecule, complicated steps had to be followed to obtain the specific optically active form. For example, according to Ger, (East) 13683 (August 27, 1957), optically active phenylacetylcarbinol was produced from benzaldehyde by .fermentation utilizing yeast and erythro-1-2-methylamino-1-phenyl-l-propanol (i.e., 1-ephedrine) could be produced by reductively condensing methylamine to the optically active phenylacetylcarbinol.
To obtain pseudoephedrine, the production is possible as described in US Patent No. 4,237,304: an oxazoline is formed from 1-ephedrine produced by the method described in Ger. (East) 13683 (August 27, 1957), using acetic anhydride, and then the oxazoline is hydrolyzed through inversion to the threo form (i.e., d-pseudoephedrine)-.
As stated above, to produce pseudoephedrine with the desired optical activity, from 2-methylamino-l-phenyl-1-propanone, steps are necessary such that ephedrine in the optical active erythro form is once produced and then it is inverted to the threo form.

2-

Hence, there arise problems that the number of steps grows and leads to complication and that the yields 1owe r.
Furthermore, in the production of the pseudoephedrine while a substantial amount of diastereomers is produced as byproducts during the reduction of the starting ketone, the recovery of the diastereomers for their use as raw material is difficult, which is economically disadvantageous.
In addition, according to the method as described in the publication of JP, 8-98697, A, it is possible to produce an optically active 2-amino-l-phenylethanol derivative from a 2-amino-l-phenylethanol compound having one asymmetric carbon atom within its molecule through the use of a specific microorganism. The present state of art is, however, that there has been no efficient process for producing a j3 -amino alcohol having two asymmetric carbon atoms. Disclosure of the Invention
This invention has been made in view of the above-indicated circumstances and it aims at producing a j3-amino alcohol having the desired optical activity from an enantiomeric mixture of an a -aminoketone compound or its salt in a high yield as well as in a highly selective manner with a simple process while
3-

sufficiently preventing the generation of diastereomeric byproducts.
The present inventors repeated studies diligently to solve the above-stated problems; consequently, it was discovered that by utilizing specific-microorganisms only one enantiomer of the enantiomeric mixture of an a-aminoketone compound or its salt could be reduced to produce the only desired kind among . the corresponding four kinds of /3 -amino alcohols in a high yield as well as in a highly selective manner. This led to the completion of the present invention.
Specifically, the process for producing an optical
active B -amino alcohol according to this invention is
a process for producing an optical active /? -amino
alcohol characterized in that it allows at least one
microorganism selected from the group consisting of
microorganisms belonging to the genus Morganella, the
genus Microbacterium, the genus Sphingobacterium, the
genus Nocardioides, the genus Mucor, the genus Absidia,
the genus Aspergillus, the genus Penicillium, the genus
Grifola, the genus Eurotium, the genus Ganoderma, the
genus Hypocrea, the genus Helicostylum, the genus
Verticillium, the genus Fusarium, , the genus
Tritirachium, the genus Mortierella, the genus Armillariella, the genus Cylindrocarpon, the genus Klebsiella, the genus Aureobacterium, the genus
5

Xanthomonas, the genus Pseudomonas, the genus Mycobacterium, the genus Sporobolomyces, the genus Sporidiobolus, the genus Amycolatopsis, the genus Coprinus, the genus Sexratia, the genus fiiiodococcus and the genus RhodotorUla to act on an enantiomeric mixture of an a -aminoketone or a salt thereof having the general formula (I):

(I)

wherein X may be the same or different and represents at least one member selected from the group consisting of a halogen atom, lower alkyl, hydroxyl optionally protected with a protecting group, nitro and sulfonyl; n represents an integer of from 0 to 3; R1 represents lower alkyl; R2 and R3 may be the same or different and represent at least one member selected from the group consisting of a hydrogen atom and lower alkyl; and "*" represents an asymmetric carbon,
to produce an optically active j3 -amino alcohol compound with the desired- optical activity having the general formula (II):
6

Further, the microorganism is preferably at least
one microorganism selected from the group consisting of
microorganisms belonging to the genus Amycolaptopsis,
the genus Coprinus, the genus Serratia, the genus
Rhodococcus and the genus Rhodotorula. More
specifically, it is preferably a microorganism selected
from the group consisting of microorganisms belonging
to Amycolatopsis alba, Amycolatopsis azurea,
Amycolatopsis coloradensis, Amycolatopsis orientalis
lurida, Amycolatopsis orientalis orientalis, Coprinus
rhizophorus, Serratia marcescens, Rhodococcus
erythropolis, Rhodococcus rhodochrous and Rhodotorula aurantiaca. By utilizing such microorganisms, (1R,2R)-amino alcohols tend to be obtained in simple processes
as the optically active j3 -amino alcohols represented by the general formula (II) in high yields as well as in a highly selective manner.
Still further, in this invention the microorganism may be cultured in a medium to which there has been added an activity inducer having the general formula
(III) :

wherein R4 represents lower alkyl; R and R may be the same or different and each represents a hydrogen atom,
,5

Fpni.nrpjoo
lower alkyl or acyl; and Y represents C=0 or CH-OH. The mediation- of such an activity inducer renders the
production of an optically active j3 -amino alcohol more
efficient.
Brief Description of the Drawings
Figs. 1A through ID are representations showing the structures of the optically active /3 -amino alcohols described below, including their absolute configurations.
Fig. 1A shows a j3 -amino alcohol with the (IS, 2S) configuration obtained according to this invention. Fig. IB shows (IS,2S)-(+)-pseudoephedrine obtained according to the invention. Fig. 1C shows a /3 -amino alcohol with the (1R, 2R) configuration obtained according to the invention. Fig. ID shows (1R, 2R)-(-)-pseudoephedrine obtained according to this invention. Best Mode for Carrying Out the Invention
The preferred embodiments of this invention will be described in detail hereafter.
The process for producing an optical active j3 -amino alcohol according to this invention is a process for producing an optical active j3 -amino alcohol characterized in that it allows at least one microorganism selected from the group consisting of microorganisms belonging to the genus Morganella, the genus Microbacterium, the genus Sphingobacterium, the

genus Nocardioides, the genus Mucor, the genus Absidia,
the genus Aspergillus, the genus Penicillium, the genus
Grifola, the genus Eurotium, the genus Ganoderma, the
genus Hypocrea, the genus tfelicostylum, the genus
Verticillium, the genus Fusarium, the genus
Tritirachium, the genus Mortierella, the genus Armillariella, the genus Cylindrocarpon, the genus Klebsiella, the genus Aureojbacteriu;n, the genus Xanthomonas, the genus Pseudomonas, the genus Mycobacterium, the genus Sporodoio^yces, the genus Sporidiobolus, the genus Afliycolatopsis, the genus CoprinuS, the genus Serratia, the genus Rhodococcus, and the genus Rhodotorula to act on an enantiomeric mixture of an a -amino ketone compound or a salt thereof having the general formula (I): ■

(r)

wherein X may be the same or different and represents at least one member selected from the group consisting of a halogen atom, lower alkyl, hydroxyl optionally protected with a protecting group, nitro and■sulfonyl; n represents an integer of from 0 to 3; R1 represents
12

lower alkyl; R2 and R3 may be the same or different and represent at least one member selected from the group consisting of a hydrogen atom and lower alkyl; and "*" represents an asymmetric carbon,
to produce an optically active j3 -amino alcohol compound with the desired optical activity having the general formula (II):

(n)

wherein X, n, R1, R2, R3 and "*" are as previously defined.
The starting material used in the process for
producing an optically active j3 -amino alcohol according to this invention is an enantiomeric mixture
of an a -aminoketone compound or a salt thereof having the general formula (I) wherein X may be the same or different and represents at least one member selected from the group consisting of a halogen atom, lower alkyl, hydroxyl optionally protected with a protecting group, nitro and sulfonyl; n represents an integer of from 0 to 3; R1 represents lower alkyl; R2 and R3 may be the same or different and represent at least one member
13

selected from the group consisting of a hydrogen atom
and lower alkyl; and "*" represents an asymmetric carbon.
The substituent group X contained in the a -amino ketone will be described in the following: the halogen atoms include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
The lower alkyl groups are preferably alkyls of
from one to six carbons and include methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl,
pentyl, isopentyl, hexyl, and the like. These may
adopt either of straight chain and branched structures
and may have as a substituent, a halogen atom such as
fluorine or chlorine, hydroxyl, alkyl, amino, Or alkoxy.
For the protecting group of the hydroxyl
optionally protected with a protecting group, there are
mentioned, among others,- the one that can be removed
upon treatment with water, the one that can be removed
by hydrogenation, the one that can be removed by a
Lewis acid catalyst or thiourea. The protecting groups
include acyl optionally having a substituent, • silyl
optionally having a substituent, alkoxyalkyl, lower
alkyl optionally having a substituent, benzyl, p-
methoxybenzyl, 2,2,2-trichloroethoxycarbonyl,
allyloxycarbonyl, trityl and the like.

The acyl groups include acetyl, chloroacetyl, dichloroacetyl, pivaloyl, benzoyl, p-nitrobenzoyl, and the like; they may also have a substituent such as hydroxyl, alkyl, alkoxy, nitro, a halogen atom, or the like. The silyl groups include trimethylsilyl, t-butyldimethylsilyl, triarylsilyl,. and the like; they may also have a substituent such as alkyl, aryl, hydroxyl, alkoxy, nitro, a halogen atom, or the like. The alkyl groups include methoxymethyl, 2-methoxyethoxymethyl and the like. The lower alkyl groups include alkyls of from one to six carbons: there are mentioned methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl, hexyl, and the like. These may adopt either of straight chain arid branched structures and may have a substituent such as a halogen atom (including fluorine and chlorine), hydroxyl, alkyl, amino,' or alkoxy.
The X may be nitro or sulfonyl, and specifically, methylsulfonyl is mentioned among others.
In addition, the number n of X is an integer of from 0 to 3, preferably 0.
R1 in the general formula (I) represents lower alkyl. Such lower alkyls are preferably alkyls of from one to six carbons: there are mentioned methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl,

pentyl, isopentyl, hexyl, and the like. These may adopt either of straight chain and branched structures. R2 and R3 represent a hydrogen atom or lower alkyl. The lower alkyls include alkyls of from one to six carbons: there are mentioned methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl, hexyl, and the like. These may adopt either of straight chain and branched structures.
The salts of the a -aminoketone compound include the salts of inorganic acids such as hydrochloride, sulfate, nitrate, phosphate, and carbonate and the salts of organic acids such as acetate and citrate.
The a -aminoketone can readily be synthesized by halogenating the a -carbon of the corresponding 1-phenyl ketone derivative (e.g., bromination) and substituting the halogen such as bromo for amine (Ger. (East) 11, 332', Mar. 12, 1956) .
The microorganism according to' this invention is
that which act on the enantiomeric mixture of the a -aminoketone having the general formula (I) or a salt thereof. Such microorganism is one selected from the group consisting of microorganisms belonging to the genus Morganella, the genus Microbacterium, the genus Sphingobacterium, the genus Nocardioides, the genus Mucor, the genus Absidia, the genus Aspergillus, the genus PeniciIlium, the genus Grifola, the genus

belonging to Amycolatopsis alba, Amycolatopsis azurea,
Amycolatopsis Coloradensis, Amycolatopsis orientalis
lurida, Amycolatopsis orientalis orientalis, Coprinus
rhizophorus, Serratia marcescens, Rhodococcus
erythropolis, Rhodococcus rhodochrous and Rhodotorula aurantiaca. By utilizing such microorganisms, (1R,2R)-amino alcohols tend to be obtained in simple processes
as the optically active j3 -amino alcohols represented by the general formula (II) in high yields as well as in a highly selective manner.
In addition, the microorganisms according to this invention include (IS,2S)-amino alcohol producing bacteria that selectively produce (1S,2S) forms among
the optically active j3 -amino alcohol compounds and (1R, 2R)-amino alcohol producing bacteria that selectively produce (1R,2R) forms among the optically
active /3 -amino alcohol compounds.
By allowing the action of the (IS, 2S)-amino
alcohol producing bacteria, there can be obtained, for
example, d-threo-2-methylamino-l-phenylpropanol(d-
pseudoephedrine), d-threo-2-dimethylamino-l-
phenylpropanol(d-methylpseudoephedrine), (IS,2S)- a -(1-aminoethyl)-benzylalcohol(d-norpseudoephedrine), (IS,2S)-1-(p-hydroxyphenyl)-2-methylamino-l-propanol, (1S,2S)- a -(1-aminoethyl)-2,5-dimethoxy-benzylalcohol, (IS,2S)-1-(m-hydroxyphenyl)-2-amino-l-propanol,

(IS,2S)-1-(p-hydroxyphenyl)-2-amino-l-propanol,
(lS,2S)-l-phenyl-2-ethylamino-l-propanol, (1S,2S)-1-
phenyl-2-amino-l-butanol and (IS,2S)-l-phenyl-2-
methylamino-1-butanol. By allowing the action of the
(IR,2R)-amino alcohol producing bacteria, there can be
obtained, for example, l-threo-2-methylamino-l-
phenylpropanol(1-pseudoephedrine), l-threo-2-
.dimethylainino~l-phenylpropanol(1-methylpseudoephedrine), (IR,2R)- a -(1-aminoethyl)-benzylalcohol(1-norpseudoephedrine), (IR,2R)-1-(p-hydroxyphenyl)-2-
methylamino-1-propanol, (IR,2R)- a -(1-aminoethyl)-2, 5-dimethoxy-benzylalcohol, (IR,2R)-1-(m-hydroxyphenyl)-2-amino-1-propanol, (IR,2R)-1-(p-hydroxyphenyl)-2-amino-l-propanol, (IR, 2R)-l-phenyl-2-ethylamino-l-propanol, (IR,2R)-l-phenyl-2-amino-l-butanol and (IR,2R)-1-phenyl-2-methylamino-l-butanol.
Additionally, the obtained (IS, 2S)-1-(m-hydroxyphenyl)-2-aminb-l-propanol can be inverted to produce (IR,2S)-1-(m-hydroxyphenyl)-2-amino-l-propanol (metaraminol).
Among the microorganisms according to this invention, those to which IFO accession numbers have been designated are described in the "List of Cultures, 10th Edition (1966)" published by Institute for Fermentation (IFO) (non-profit organization) and are available from the IFO. The microorganisms to which

IAM accession numbers have been designated are described in the "Catalogue of Strains, 1993" published by Institute of Molecular and Cellular Biosciences, the Cell & Functional Polymer General Center, University of Tokyo and are available from its preservation facilities. Further, Rhodococcus erythropolis MAK-34 is a novel microorganism isolated from the nature and has been deposited with National Institute of Bioscience and Human-Technology, National Institute of Advanced Science and Technology, METI locating at 1-3, Higashi 1-Chome, Tsukuba, Ibaraki, JAPAN (postal code: 305-8566) as FERM BP-7451 (the date of original deposit: February 15, 2001) .
For the microorganism used in this invention, there can be used any of wild-type strains, mutant strains and -recombinant strains derived by the techniques of cell .engineering such as cell fusion or by the techniques of genetic engineering such as gene manipulations insofar as it is a microorganism capable of acting on the enantiomeric mixture of a a -aminoketone compound of the general formula (I) and producing the corresponding optically active j3 -amino alcohol of the general formula (II).
There are no particular limitations to the different conditions in the culturing of the microorganisms, and the methods that are ordinarily

33-

used may be carried out, where bacteria, fungi, and yeast are cultured in suitable media, respectively. Normally, there may be used liquid media containing carbon sources, nitrogen sources and other nutrients. Any sources may be used for the carbon source of the medium as long as the microorganisms can utilize them. Specifically, there may be used sugars such as glucose,' fructose, sucrose, dextrin, starch, and sorbitol; alcohols such as methanol, ethanol, and glycerol; organic acids such as fumaric acid, citric acid, acetic acid, and propionic acid and their salts; hydrocarbons such as paraffin; and mixtures of the foregoing. Any sources may be used for the nitrogen source of the medium as long as the microorganisms pan utilize them. Specifically, there may be used the ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, and ammonium' phosphate; the ammonium salts of organic acids such as ammonium fumarate and ammonium citrate; the salts of nitric acid such as sodium nitrate and potassium nitrate; nitrogen-containing inorganic or organic compounds such as beef extract, yeast extract, malt extract, and peptone; and mixtures of the foregoing. Nutrition sources may also be added appropriately to the medium, which are used in the normal culturing, including inorganic salts, the salts of minute metals, and vitamins. There may also be

added to the medium, a substance for inducing the activity of a microorganism, a buffer substance effective to maintain pH, or the like.
The substances for inducing the activity of a microorganism include an activity, inducer having the general formula (III) :
/R6
4/Y\/\ (IE)
R4 V
wherein R4 represents lower alkyl; Rs and R6 may be the same or different and each represents a hydrogen atom, lower alkyl, or acyl; Y represents C=0 or CH-OH.
The lower alkyl and acyl groups include the ones
previously defined respectively. Specifically, the
preferred activity inducers include l-amino-2-propanol,
l-amino-2-hydroxybutane, l-acetylamino-2-propanol, 1-
methylamino-2-propanol, l-amino-2-oxopropane, 2-amino-
3-hydroxybutane, and the like. When asymmetric carbons
are present in these compounds, the compounds may be
either of optically .active forms and racemates, and may
appropriately be selected. The addition of these
activity inducers to medium induces the activity of
microorganisms and the subsequent generation of
optically active j3 -amino alcohols to progress with
higher efficiency as compared to the case with no such
addition. The activity inducers may be used

individually, or may be used as a mixture of plural inducers. The addition levels of such activity inducers are desirably 0.01 to 10 wt. % relative to medium.
Culturing of microorganisms can be carried out under the conditions suited for their growth. Specifically, it can be done at the pH of medium being 3-10, preferably pH 4-9 and at a temperature of 0-50 C, preferably 20-40 ° C. The culturing of .S microorganisms can be carried out under aerobatic conditions or anaerobatic conditions. The culturing time is preferably from 10 to 150 hours and should be appropriately determined for the respective tmicroorganisms.
The reaction method in the- production of j3 -amino alcohols according to this invention is not particularly limited insofar as it is a method by which the microorganism acts on the enantiomeric mixture of the a-amino ketone compound having the general formula (I) or a mixture thereof to produce the corresponding
optically active |3 — amino alcohol compound having the general formula (II). The reaction is allowed to start by mixing bacterial cells washed with buffer or water to a aqueous solution of the starting a-aminoketone.
The reaction conditions can be selected, from the range within which the generation of the optically

•25-

active j3 -amino alcohol compound having the general formula (II) is not impaired. The quantity of bacterial cell is preferably 1/100 to 1000 times, and more preferably 1/10 to 100 times that of racemic aminoketone. The concentration of the racemic aminoketone that is a substrate is preferably from 0.01 to 20%, and more preferably from 0.1 to 10%. Further, the pH of reaction solution is preferably from 5 to 9, and more preferably from 6 to 8; the reaction temperature is preferably from 10 to 50 ° C, and more preferably from 20 to 40 ° C. Still further, the reaction time is preferably from 5. to 150 hours and it should be appropriately determined for the respective microorganisms.
In order for the reaction to progress more efficiently, sugars (e.g., glucose), organic acids (e.g., acetic acid), and energy substances (e.g., glycerol) may be added. These may.be respectively used alone or may be used as a mixture thereof. The level of addition is preferably 1/100 to 10 times that of substrate. Coenzymes or the like may also be added. Coenzymes such as nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) can be used alone or as a mixture of the
27

foregoing. The level of addition is preferably from 1/1000 to 1/5 times that of the racemic aminoketone. In addition to these coenzymes coenzyme-regenerating enzymes such as glucose dehydrogenase may also be added; and the level of addition is preferably from 1/100 to 10 times that of the racemic aminoketone. Further, sugars (e.g., glucose), organic acids (e.g., acetic acid), and energy substances (e.g., glycerol), coenzymes, coenzyme-regenerating enzymes, and substrates for the coenzyme-regenerating enzymes may respectively be combined for use. These substances are naturally accumulated in bacterial cells, but where their addition as required can increase the reaction rate and yield, they may be appropriately selected.
Furthermore, when a certain salt is added such that the reaction solution may. be as described above and the reaction solution is allowed to react under that condition, the racemization of the unreacted a -aminoketone isomer can be accelerated and its conversion to the enantiomer that serves as the substrate for microorganism can be progressed more efficiently. 'This tends to produce the objective amino alcohol in a high yield of 50% or more from the starting material.
The salts for accelerating the racemization of the unreacted a -aminoketones may be the salts of weak
28

acids such as acetate, tartrate',, benzoate, citrate, malonate, phosphate, carbonate, p-nitrophenolate, sulfite, and borate. Preferably, there are used phosphates (e.g., sodium dihydrogenphosphate, potassium dihydrogenphosphate, and ammonium dihydrogenphosphate), carbonates (e.g., sodium carbonate, sodium hydrogencarbonate, potassium carbonate, and ammonium carbonate), citrates (e.g., sodium citrate, potassium citrate, and ammonium citrate), for example. These mixtures may also be used, and desirably, buffers with pH 6.0-8.0 are added to give final concentrations of from 0.01 to 1 M. For example, in the case of a phosphate, sodium dihydrogenphosphate and sodium monohydrogenphosphate are desirably mixed at a ratio of from 9:1 to 5:95.
The optically active a-amino alcohols produced by
reaction may be purified by conventional
separation/purification means. For example, directly
from the reaction solution or after the bacterial cells
are separated, optically active j3 -amino alcohols can
be obtained by being subjected to normal purification
methods such as membrane separation or extraction with
an organic solvent (e.g., toluene and chloroform),
column chromatography, concentration at reduced
pressure, distillation, recrystallization, and
crystallization.
29

GCfll 0025 00-
The optical activity of the optically active /3 -amino alcohol thus produced can be determined by high performance liquid chromatography (HPLC). EXAMPLES
This invention will be described concretely by way of examples; however, the scope of the invention is not to be limited by these examples.
(Preparation Example 1) Preparation of dil¬ute thylamino-1-phenyl-1-propanone
Bromine (51.6 ml) was added dpapwioo-to a mixture of 1-phenyl-l-propanone (134 g) , sodium carbonate (42 g) and water (200 ml), and reaction was allowed to take
place at 70 ° C for 3 hours to give a reaction mixture. To the reaction mixture was added 40% aqueous monomethylamine solution (350 ml) . After allowing to react at 40 ° C for 1 hour, the reaction product was extracted into chloroform (11) . The reaction product in'the chloroform layer was then extracted with dilute hydrochloric acid (100 ml), and" activated carbon (3 g) was added to the aqueous layer and filtrated. The filtrate was concentrated to give dl-2-methylamino-l-
t-
phenyl-1-propanojue—Jiydjrochloride (89 9) •
(Example 1) Production of d-(IS,2S)-pseudoephedrine
Microbacterium arborescens IFO 3750 was inoculated to a medium (5 ml) containing 1% glucose, 0.5% peptone, and 0.3% yeast extract, and shake-culturing was carried
30

out at 30 ° C for 48 hours. After the cultured solution was centrifuged to give bacterial cells, the cells were placed into a test tube. To this was added 0.1 M sodium phosphate buffer (pH 7.0, 1 ml) and suspended. To this was added dl-2-methylamino-l-phenyl-1-propanone hydrochloride (1 mg) and reaction was allowed to take place under shaking at 30 ° C for 24 hours. After the reaction, the reaction solution was centrifuged to remove the bacterial cells and the supernatant was subjected to HPLC to give optically
active pseudoephedrine: p. Bondapakphenyl manufactured by Waters Inc.; diameter of 4 mm; length of 300 mm; eluent-0.05 M sodium phosphate buffer (containing 7% acetonitrile) ; pH 5.0; flow rate of 0.8 ml/min; and detection light wavelength at 220 nm.
The absolute configuration and optical purity were determined with HPLC (Column Sumichiral AGP manufactured by ■Sumika Chemical Analysis Service; diameter of 4 mm; length of 150 mm; 0.03 M sodium phosphate buffer; pH 7,0; flow rate of 0.5 ml/min; and detection light wavelength at 220 nm) . ■ Consequently, only d-pseudoephedrine was obtained selectively, as shown in Table 1.
The produced amounts will be all shown in terms of the amounts of converted hydrochloride hereafter.

(Examples 2 to 12) Production of d-(lS,2S)-pseudoephedrine
Except that the microorganisms shown in Table 1 were used in place of Microbacterium arborescens IFO 3750, optically active pseudoephedrine was obtained similarly to Example 1. Consequently, only d-
pseudoephedrine was obtained selectively, as shown in Table 1.
Table 1

Except that the microorganisms shown in Table 2 were used in place of Microbacterium arborescens IFO 3750, optically active pseudoephedrine was obtained similarly to Example 1. The produced amounts and
optical purities of pseudoephedrine are shown In Table 2.
Table 2

Morganella morganii IFO 3848 was inoculated to a medium containing 1% glucose, 0.5% peptone, and 0.3% yeast extract, and shake-culturing was aerobically carried out at 30 ° C for 48 hours. After this cultured solution (5 ml) was centrifuged to give bacterial cells, the cells were dried in the air and the resulting dried bacterial cells were suspended in 1 ml of 0.05 M Tris hydrochloric acid buffer (pH 7.5). To the aforementioned dried bacterial cell suspension were added glucose (50 mg) , glucose dehydrogenase (0.2 mg), NADP (0.6'mg), NAD (0.6 mg), and dl-2-methylamino-1-phenyl-l-propanone hydrochloride (10 mg) and
reciprocation-shaking was carried out at 28 ° C and at 300 rpm. After allowing to react for 48 hours, the reaction solution was measured for the produced amount and the optical activity of pseudoephedrine by HPLC similarly to Example 1. Consequently, only d-. pseudoephedrine was obtained selectively, as shown in Table 3.
Table 3

Mycobacterium smegmatis IAM-12065 was inoculated to a medium containing 1% glucose, 0.5% peptone, and 0.3% yeast extract, and shake-culturing was aerobically carried out at 30 ° C for 48 hours. After the cultured solution (1 1) was centrifuged to give bacterial cells, the cells was suspended in 50 ml of water, and after addition of dl-2-methylamino-l-phenyl-l-propanone hydrochloride (0.5 g) , reciprocation-shaking was carried out at 30 ° C and at 150 rpm. One hundred hours after the start of shaking, 7.0 g/1 of d-pseudoephedrine was produced in the reaction solution. After the reaction solution was centrifuged to remove the bacterial cells, the pH was adjusted to 12 or greater by addition of sodium hydroxide. Methylene chloride (100 ml) was added to this reaction solution and the reaction product was extracted. The solvent was removed, hydrochloric acid was added, and then concentration to dryness yielded a hydrochloride salt. The hydrochloride salt was dissolved by addition of ethanol and further addition of ether crystallized the reaction product. Consequently, d-pseudoephedrine hydrochloride was obtained. The resulting d-pseudoephedrine crystals (0.32 g) were analyzed on HPLC (Column Sumichiral AGP'manufactured by Sumika Chemical Analysis Service; diameter of 4 mm; length of 150 mm; 0.03 M sodium phosphate buffer; pH 7.0; flow rate of

35

■f¥e±-ee2s«Qa_
0.5 ml/min; detection light wavelength at UV 220 nra) and the optical activity was found to be 100%.
(Example 40) Production . of d-(lS,2S)-
methylpseudoephedrine
Mycobacterium smegmatis IAM-120 65 was inoculated to a medium containing 1% glucose, 0.5% peptone, and 0.3% yeast extract, and shake-culturing was aerobically carried out at 30 ° C for 48 hours. The cultured solution (1 1) was filtrated to give bacterial cells, the resulting cells were washed wi.th Water, and water was added to form 50 ml of suspension. To the suspension was added 100 mg of 2-dimethylamino-l-phenyl-1-propanone hydrochloride (2 g/1), and
reciprocation-shaking was carried out at 30 ° C and at 150 rpm for 48 hours. When the reaction solution was analyzed on HPLC (Column Sumichiral AGP manufactured by Sumika Chemical Analytical Center; diameter of 4 mm; length of 150 mm; 0.03 M sodium phosphate buffer; pH 7.0; flow rate of 0.5 ml/min; detection light wavelength at UV 220 nra), it was found that d-(lS,2S)-methylpseudoephedrine was produced at 0.23 g/1 and its optical activity was 77%. (Example 41) Production of 1-(1R,2R)-pseudoephedrine
Amycolatopsis alba IFO 15602 was inoculated to a medium containing 1% glucose, 0.5% peptone and 0.3% yeast extract, and shake-culturing was aerobically
36

carried out at 30 ° C for 48 hours. The cultured solution (5 ml) was centrifuged to give bacterial cells After the cells were suspended in 1 ml of 0.1 M sodium phosphate (pH 7.0) and dl-2-methylamino-l-phenyl-l-propanone hydrochloride (1 mg) was added thereto, reaction was allowed to take place by carrying out reciprocation-shaking at 30 ° C and at 150 rpm for 48 hours. When the reaction solution was analyzed on HPLC (Column Sumichiral AGP manufactured by Sumitomo Chemical Analytical Center Co. Ltd.; diameter of 4 mm; length of 150 mm; 0.03 M sodium phosphate buffer; pH 7.0; flow rate of 0.5 ml/min; detection light wavelength at UV 220 nm), it was found that 1-pseudoephedrine was produced selectively. The result is shown in Table 4.
(Examples 42 ■ to 46) Production of 1-(1R,2R)-pseudoephedrine
Except that the microorganisms shown in Table 4 were used in place of Amycolatopsis alba IFO 15602, optically active pseudoephedrine was obtained similarly to Example 41. Consequently, only 1-pseudoephedrine was obtained selectively, as shown in Table 4.
Table 4

.EperotEOT'
1M phosphate buffer (0.2 ml; pH 7.0), glucose (10 mg) and racemic l-'(p-hydroxyphenyl) -2-methylamino-l-propanone hydrochloride (1 mg) , and they were mixed.. One milliliter was 'shaken for reaction at 30 ° C for 48 hours. The reaction solution was either centrifuged or filtered. The supernatant was analyzed on HPLC (p. Bondapakphenyl manufactured by Waters Inc.; diameter of 4 mm; length of 300 mm;' eluent-0.05 M sodium phosphate buffer (containing 7% acetonitrile).; pH 6.5; flow rate of 0.8 ml/min; detection wavelength at UV 220 nm). Consequently, it was confirmed that threo-l-(p-hydroxyphenyl)-2-methylamino-l-propanol hydrochloride was produced at 0.6 mg/ml. To determine the optical purity of the product, the sample was analyzed on HPLC (Sumichiral OA-4 900 manufactured by Sumika Chemical Analysis Service;
eluent :hexane:dichloroethane .-methanol: trifluoroacetic acid=240:140:40:1; flow rate of 1 ml/min; detection wavelength at UV 254 nm). Consequently, it was found that (1S,2S)-1-(p-hydroxyphenyl)-2-methylamino-l-propanol was obtained in 100% optical purity. (Examples 51 to '54) Production of optically active 1-(p-hydroxyphenyl)-2-methylamino-l-propanol
Except that the microorganisms shown in Table 7 were used in place of Rhodococcus erythropolis MAK-34 strain and the culturing conditions in the table were
40

followed, reaction was carried out similarly to Example 50 and optically active 1-(p-hydroxyphenyl)-2-methylamino-1-propanol was obtained. The results are shown in Table 7. In all instances, the optically active compound was obtained efficiently.
The culture conditions listed in Tables 7-9 are as follows:
Culture conditions 1: A microorganism was inoculated to a medium (20 ml) containing 1% glucose, 0.5% peptone, and 0.3% yeast extract (pH7.0) and culturing was
carried out at 30 ° C' for 48 hours under the shaking condition of 150 rpm.
Culture conditions 2: A microorganism was inoculated, to a medium (20 ml, pH 6.0) containing 5% malt extract and 0.3% yeast extract and culturing was carried out at
30 ° C for 48 hours under the shaking condition of 150 rpm.
Table 7

FP01-UU25--O0
i.
1 ml/min; detection wavelength at UV 254 nm).
Consequently, the product was found to be (lS,2S)-2-
ethylamino-1-phenyl-l-propanol (optical purity: 100%) .
(Examples 56 to 58) Production of (1R,2R)-2-ethylamino-
1-phenyl-1-propanol
Except that the microorganisms shown in Table 8 were used in place of Rhodococcus erythropolis MAK-34 strain and the culturing conditions in the table were followed, reaction was carried out similarly to Example 55 and (1R,2R)-2-ethylamino-l-phenyl-l-propanol was obtained. The results are shown in Table 8.
Table 8

Amycolatopsis alba IFO-15602 was cultured under culture conditions 1, and either centrifugation or filtration yielded bacterial cells. To these were added an adequate amount of water, 1M phosphate buffer (0.2 ml, pH 7.0), glucose (10 mg) and racemic l-(p-hydroxyphenyl)-2-amino-l-propanone hydrochloride (1 mg), and they were mixed. One milliliter was shaken for reaction at 30 ° C for 48 hours. This was either centrifuged or filtered. The supernatant was analyzed
on HPLC ( p, Bondapakphenyl manufactured by Waters Inc.; diameter of 4 mm; length of 300 mm; eluent-0.05 M sodium phosphate buffer (containing 7% acetonitrile); pH 6.5; flow rate of 0.8 ml/min; detection wavelength at UV 220 nm) . Consequently, it was confirmed that t/ireo-1- (p-hydroxyphenyl) -2-amino-l-propanol hydrochloride was produced at 0.03 mg/ml. To determine the optical purity of the product, the sample was analyzed on HPLC (Crownpak, CR+ manufactured by Uaicel Chemical Industries Ltd.; perchloric acid, pH 2.0, 1.0 ml/min, UV 254. nm) . Consequently, the product was found to be (1R, 2R)-1-(p-hydroxyphen,yl)-2-amino-l-propanol (optical purity: 82%).
(Example 63) Production of (IS,2S)-l-phenyl-2-amino-l-butanol
Helicostylum nigricans IFO-8091 was cultured under culture ■ conditions 1. Either centrifugation or
45

filtration yielded bacterial cells. To these were
added an adequate amount of water, 1M phosphate buffer
(0.2 ml, pH 7.0), glucose (10 mg) and racemic 1-phenyl-
2-amino-l-butanone hydrochloride (1 mg) , and they were
mixed. One milliliter was shaken for reaction at 30 C for 48 hours. The reaction solution was either centrifuged or filtered. The supernatant was analyzed on HPLC ( ix Bondaspherephenyl manufactured by Waters Inc.; diameter of 4 mm; length of 150 mm; eluent-0.05 M sodium phosphate buffer (containing 7% acetonitrile); pH 6.5; flow rate of 0.8 ml/min; detection wavelength at UV 220 nm) . Consequently, it was found that threo-l-phenyl-2-amino-l-butanol hydrochloride was produced at 0.62 mg/ml. To determine the optical purity of product, the sample was analyzed on HPLC (OD by Daicel Chemical Industries Ltd.; diameter of 4.6 mm; length of 250 mm; hexane:isopropanol:diethylamine=90:10:0.1; 1 ml/min; UV 254 nm) . ' Consequently, the product was found to be (IS,2S)-l-phenyl-2-amino-l-butanol. (Example 64) Production of (1R,2R)-l-phenyl-2-amino-l-butanol
Amycolatopsis orientalis IFO-12806 was cultured under culture conditions 1, and similarly to Example 63, (1R, 2R)-l-phenyl-2-amino-l-butanol could be produced at 0.21 mg/ml. (Example 65) The effect of addition of inducer (1)
46

l-Amino-2-hydroxypropanone was added to medium 1 (Table 10) so that a level of 5g/L could be obtained. Five milliliters was then poured into' a test tube. With a silicone stopper it was sterilized in an autoclave at 121. °C for 30 minutes. The microorganisms listed in Table 11 were inoculated to this medium and to the medium with no addition of the inducer, respectively; and they were shake-cultured at 300 rpm
and at 30 ° C for 48 hours. The culture (0.5 mL) was centrifuged at 10,000 G for 20 minutes. The bacterial cells obtained by removal of the supernatant were suspended by addition of water to prepare a uniform suspension. To this were added water, buffer and dl-2-methylamino-1-phenyl-l-propanone hydrochloride (10 mg) , forming 1 mL. The one milliliter was poured into a test tube and reaction was allowed to take place under shaking at 150 rpm and at 30 ° C for 12 hours. After the reaction, the bacterial cells were removed by centrifugation and the supernatant was subjected to HPLC, whereby the produced amount of pseudoephedrine was determined (HPLC conditions: a Bondapakphenyl manufactured by Waters Inc.; diameter of 4 mm; length of 300 mm; eluent-0.05 M sodium phosphate buffer (containing 7% acetonitrile); pH 6.5; flow rate of 0.8 ml/min; detection wavelength at UV 220 nm).
47

As the results are shown in Table 11, the produced amounts of pseudoephedrine when culturing was carried out with the addition of the inducer displayed remarkable increases as compared to the culturing with no addition of inducer.
Table 10

Compound name Produced amount (nig)
l-acetylamino-2-propanol 3.00
l-methylamino-2-propanol 2.83
l-amino-2-oxopropane 1.97
2-amino-3-hydroxybutane 0.05
l-amino-2-hydroxybutane 0.65
no addition 0.02
(Comparative Example 1)
Except that Brettanomyces anomalus IFO 0642 was used instead of Microbacterium arborescens IFO 3750, the production reaction of pseudoephedrine was attempted similarly to Example 1. However, no reduced product was obtained. (Comparative Example 2)
Except that Candida guilliermondii IFO 05 66 was used instead of Microbacterium arborescens IFO 3750, the production reaction of pseudoephedrine was attempted similarly to Example 1. However, no reduced product was obtained. (Comparative Example 3)
Except that Schizosaccharomyces pombe IFO 0358 was used instead of. Microbacterium arborescens IFO 3750, the production reaction of pseudoephedrine was attempted similarly to Example 1. However, no reduced product was obtained. (Comparative Example 4) ' .

Except that Bacillus subtilis IFO 3037 was used instead of Microbacterium arborescens IFO 3750, the production reaction of pseudoephedrine was attempted similarly to Example 1. However, no reduced product was obtained. Industrial Applicability
As described above, the process for producing an optically active j3 -amino alcohol according to this invention allows the j3 -amino alcohol having the desired optical activity to be produced from an
enantiomeric mixture of an a-aminoketone compound or a salt thereof in a high yield as well as in a highly selective. manner with a simple process while sufficiently preventing the generation of diastereomeric byproducts.
Accordingly, this invention will make it possible to produce pseudoephedrines, among others, having the desired optical activity in' a high yield as well as in a highly selective manner and thus it is valuable in the manufacture of drugs and their intermediates.
-51-

WE CLAIM:
A process for producing an optically active (J-amino alcohol, comprising a step of culturing a microorganism in a medium with a pH of 3 to 10 and at a temperature of 0°C to 50°C, containing an enantiomeric mixture of an a-amino ketone or a salt thereof having the general formula (I):

(n)

to produce, an optically active 0-amino alcohol compound with the desired optical activity having the general formula (II) :

wherein R4 represents lower alkyl; R5 and R6 may be the same or different and each represents a hydrogen atom, lower alkyl or acyl; and Y represents C=0 or CH-OH.
Dated this 25th day of September, 2002.
[RANJNATtfEHTA-DUTT]
OF REMFRY & SAGAR
ATTORNEY FOR THE APPLICANTS